The present invention relates to recombinant DNA comprising DNA coding for myosin heavy chain SM1 isoform protein inserted into vector DNA, a microorganism carrying the recombinant DNA and an agent for treatment of arteriosclerosis comprising the recombinant DNA which are used in gene therapy.
Smooth muscle-type myosin heavy chain SM1 isoform protein is responsible for contraction and relaxation of smooth muscles, and is one of the myosin heavy chain isoform proteins expressed specifically in smooth muscle cells. As the DNA coding for the protein, the nucleotide sequence of cDNA coding for rabbit SM1 isoform is known (P. Babij et al.: Proc. Natl. Acad. Sci. USA, 88, 10676 (1991)), but there is not known any homology among nucleotide sequences for such DNAs. Further, there is not known any recombinant DNA comprising DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein inserted into vector DNA which recombinant DNA can be injected into animal cells.
It has been reported that when cDNA coding for a protein called calponin which is a protein existing in smooth muscle cells is introduced and expressed in a vascular smooth muscle cell line derived from rat pulmonary arteries, the time required for doubling the cells is prolonged by about 4 hours (Takahashi et al.: Circulation, 88, I-174 (1993)) and that when cDNA for human calponin is injected into a rabbit topically at a site where the carotid artery has been abraded with a balloon, thickening of the intima is inhibited (Takahashi et al.: Circulation, 88, I-656 (1993)). It is not known that recombinant DNA comprising DNA coding for myosin heavy chain SM1 isoform protein inserted into vector DNA has pharmacological effect and is used for in gene therapy.
For treatment of arteriosclerosis, percutaneous transluminal coronary angioplasty (PTCA) in which a stenosed site is enlarged by a balloon catheter is extensively conducted. The treatment method has the advantages of easier operation and higher degree of success than the bypass surgery etc., while the treatment method has the disadvantage that 30 to 40% of the patients after the treatment suffer from restenosis of blood vessels due to abnormal proliferation of vascular cells. Therefore, there is demand for developments in an agent for treatment of arteriosclerosis effectively used for preventing such restenosis.
The present invention includes:
(1) A recombinant DNA comprising DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein inserted into vector DNA.
(2) The recombinant DNA according to (1) wherein the vector DNA is a retrovirus vector, adenovirus vector, adeno-associated virus vector or a plasmid capable of being expressed in an animal.
(3) The recombinant DNA according to (2) wherein the plasmid capable of being expressed in an animal is pCXN2 or PAGE208.
(4) The recombinant DNA according to any one of (1) to (3) wherein the smooth muscle-type myosin heavy chain SM1 isoform protein is of a human smooth muscle type, rabbit muscle type or mouse muscle type.
(5) The recombinant DNA according to any one of (1) to (3) wherein the DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein is the nucleotide sequence shown in SEQ ID NO:1, or the nucleotide sequence shown in SEQ ID NO:1 in which at least one nucleotide is added, deleted or replaced.
(6) DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein, which is the nucleotide sequence shown in SEQ ID NO:1, or the nucleotide sequence shown in SEQ ID NO:1 in which at least one nucleotide is added, deleted or replaced.
(7) A microorganism carrying the recombinant DNA of (5).
(8) The microorganism according to (7) which belongs to the genus Escherichia.
(9) An agent for treatment of arteriosclerosis which comprises the recombinant DNA of any one of (1) to (5).
The DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein includes cDNA or genomic DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein, preferably DNA derived from humans, rabbits, mice, etc.
The nucleotide sequence of the DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein includes the nucleotide sequence of cDNA (SEQ ID NO:5) coding for rabbit SM1 isoform protein (SEQ ID NO:4) (Proc. Natl. Acad. Sci. USA, 88, 10676 (1991)), the nucleotide sequence of the DNA coding for mouse myosin heavy chain SM1 isoform protein as shown in SEQ ID NO:1, and the nucleotide sequence as shown in SEQ ID NO:2 which is specified by homologous regions between the above nucleotide sequences (In the Sequence Listing, Y represents T, U or C; S represents G or C; V represents A, G or C; B represents B, C, T or U; W represents A, T, or U; N represents A, C, G, T, U or a single bond; and the codon YAS (388-390) represents TAC or CAG.). Accordingly, the nucleotide sequence shown in SEQ ID NO:2 contains the nucleotide sequence of SEQ ID NO:1 and the nucleotide sequence of the cDNA coding for rabbit SM1 isoform protein. A partial nucleotide sequence of cDNA coding for human smooth muscle-type myosin heavy chain SM1 isoform protein is also known (Amer. J. of Medical Genetics, 46, 61-67 (1993)), and a nucleotide sequence of this partial nucleotide sequence combined with a partial nucleotide sequence of SEQ ID NO:2 is also contained in the nucleotide sequence of the DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein according to the present invention.
Further, the nucleotide sequence of SEQ ID NO:2 or the combination of a partial nucleotide sequence of SEQ ID NO:2 and the nucleotide sequence of the DNA derived from human smooth muscles, in which one or more nucleotides have been added, deleted or replaced by means of site-directed mutagenesis (Nucleic Acid Research, 10, 6487-6508 (1982)), is also contained in the nucleotide sequence of the DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein according to the present invention.
The vector DNA that can be used includes virus vectors such as retrovirus vector, adenovirus vector and adeno-associated virus vector and plasmids capable of being expressed in an animal such as pCXN2 (Gene, 108, 193-200 (1991)) and PAGE207 (Japanese Patent Laid-Open Publication No. 46841/1994) as well as their modified vectors.
The agent for treatment of arteriosclerosis according to the present invention can be produced by compounding the recombinant DNA comprising DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein inserted into vector DNA as an active ingredient together with a base used in agents for gene therapy. Just before administration, the pharmaceutical preparation can be used in gene therapy for arteriosclerosis, if necessary after encapsulation in liposomes etc. (Proc. Natl. Acad. Sci. USA., 90, 11307 (1993)).
Where the DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein is inserted into a virus vector, a therapeutic agent can be produced by preparing virus particles containing the recombinant DNA and then compounding them together with a base used in agents for gene therapy (Nature Genet., 8, 42(1994)).
The base used in agents for gene therapy may be any base generally used in injections. The base includes, for example, distilled water, a salt solution of sodium chloride, a mixture of sodium chloride and an inorganic salt, or the like, solutions of mannitol, lactose, dextran, glucose, etc., solutions of amino acid such as glycine, arginine, etc., a mixed solution of an organic acid solution or a salt solution and glucose solution, and the like. Further, injections may be prepared in a usual manner as a solution, suspension or dispersion by adding adjuvant such as an osmotic pressure controlling agent, pH adjusting agent, vegetable oils such as sesame oil, soybean oil, etc. or surface active agents such as lecithin, non-ionic surface active agent, etc. to the above base. These injections can also be powdered, lyophilized, etc. to be dissolved just before use.
The agent for treatment of arteriosclerosis can be used as such in the case of a solution, and in the case of a solid it is dissolved in the base previously sterilized if necessary just before use in gene therapy.
The method for administration of the agent for treatment of arteriosclerosis according to the present invention involves topically administrating it into a patients who underwent the PTCA treatment, at a dose of 1 ng to 1 g per day or once after the surgery by means of a catheter etc. such that it can be absorbed into their vascular smooth muscle cells at the target site.
The agent for treatment of arteriosclerosis according to the present invention is safe in this dose range.
Hereinafter, the process for producing the recombinant DNA comprising DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein inserted into vector DNA is described.
The recombinant DNA comprising DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein inserted into vector DNA can be obtained according to the usual genetic engineering method described below or its modified method.
A DNA clone coding for smooth muscle-type myosin heavy chain SM1 isoform protein is detected in a cDNA library from tissues composed mainly of smooth muscles in the uterus, aorta, etc., according to the method described by A. Abe et al. (ECL direct DNA labeling detection system manual (Amersham)).
The recombinant DNA comprising the inserted DNA coding for myosin heavy chain SM1 isoform protein can be produced by ligating said DNA fragment to a downstream region from a promoter in a suitable vector DNA (J. Sambrook et al., Molecular Cloning, 2nd Ed., Vol. 1, Cold Spring Harbor Laboratory Press (1989)). Animal cells are used as a host, and a promoter derived from SV40, a promoter from retrovirus, a metallothionein promoter, xcex2-actin promoter etc. can be utilized as the promoter. For the expression, the use of an enhancer is also effective.
The reaction conditions for the above-mentioned recombinant techniques are as follows: The digestion of DNA with a restriction enzyme is carried out by allowing the restriction enzyme in an amount of 0.1 to 100 units, preferably 1 to 3 units per xcexcg DNA to act on 0.1 to 20 xcexcg DNA in a reaction solution usually containing 2 to 200 mM preferably 10 to 40 mM Tris-HCl buffer, pH 6.0 to 9.5, preferably pH 7.0 to 8.0, 0 to 200 mM sodium chloride, and 2 to 20 mM preferably 5 to 10 mM magnesium chloride, at 20 to 70xc2x0 C. (the optimum temperature is varied depending on the restriction enzyme used) for 15 minutes to 24 hours. The termination of the reaction can be effected usually by heating the reaction solution at 55 to 75xc2x0 C. for 5 to 30 minutes or by inactivating the restriction enzyme with reagents such as phenol etc. The DNA fragment, or the gapped duplex DNA, generated by digestion with the restriction enzyme can be purified using Prep-A-Gene Matrix (Bio-Rad). The ligation of the DNA fragment can be effected using a DNA ligation kit (Takara Shuzo Co., Ltd.).
The recombinant DNA containing DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein thus constructed in this manner is used to produce a transformant.
The host for the plasmid capable of being expressed in an animal includes, for example, microorganisms belonging to the genus Escherichia, such as Escherichia coli K12xc2x7HB101 (H. W. Boyer et al.: J. Mol. Biol.,41, 459 (1969)), DH5 xcex1 (D. Hanahan: J. Mol. Biol., 166, 557 (1983)) etc. The transformation of microorganisms of the genus Escherichia can be effected according to the method of Cohen et al. (S. N. Cohen et al.: Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)).
The host used for the virus vector includes animal cells having the ability to produce viruses, such as monkey cell COS-7, Chinese hamster cell CHO, mouse cell BALB/3T3, human cell HeLa, etc.; the host for the retrovirus vector includes xcexa8CRE, xcexa8CRIP (Proc. Natl. Acad. Sci. USA, 85, 6460 (1988)), MLV (J. Virol., 65, 1202 (1991)) etc.; and the host for the adenovirus vector and adeno-associated virus vector includes 293 cells derived from human fetal kidney (xe2x80x9cjikken Igakuxe2x80x9d (Experimental Medicine), 12, 316 (1994)) etc. The introduction of the virus vector into animal cells can be effected using the calcium phosphate method (Virology, 52, 456 (1973)) etc.
The resulting transformant can be cultured in the following manner depending on the difference in the cell species to produce the recombinant DNA.
To culture the transformant from a microorganism of the genus Escherichia as the host, the suitable medium is a liquid medium containing a carbon source, nitrogen source, inorganic substance, etc. necessary for the growth of the transformant. The carbon source includes e.g. glucose, dextrin, soluble starch, sucrose, glycerol, etc. ; the nitrogen source includes e.g. an ammonium salt, peptone, casein, etc.; and the inorganic substance includes e.g. calcium chloride, sodium dihydrogenphosphate, magnesium chloride, etc. A yeast extract, vitamins, etc. may further be added. The pH of the medium is preferably about 5 to 8. To culture the microorganism of the genus Escherichia, the medium is preferably Terrific broth (K. D. Tartof et al.: Bethesda Res. Lab. Focus, 9, 12 (1987)) or the like. The transformant is cultured usually at about 15 to 43xc2x0 C. for about 8 to 24 hours, if necessary under aeration or stirring. After the culture is finished, the recombinant DNA containing DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein can be obtained through purification by the Birnboim method (Nucleic Acid Res., 7, 1513 (1979)), etc.
To culture the transformant from animal cells as the host, the medium used includes a medium containing about 5 to 20% fetal bovine serum, such as 199 medium (Morgan et al.: Proc. Soci. Biol. Med., 73, 1 (1950)), MEM medium (H. Eagle: Science, 122, 501 (1952)), DMEM (R. Dulbecco et al.: Virology, 8, 396 (1959)) or the like. The pH is preferably in the range of about 6 to 8. The transformant is cultured usually at about 30 to 40xc2x0 C. for about 18 to 60 hours, if necessary under aeration or stirring.
Because virus particles containing the recombinant DNA are released into the culture supernatant, the recombinant DNA containing DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein can be obtained from the supernatant by concentrating and purifying virus particles by the cesium chloride centrifugation method (Koji Sawada et al.: xe2x80x9cShin Seikagaku Jikken Kouza 2-Vxe2x80x9d (New Biochemistry Experimental Course 2-V), 33 (1992)), the polyethylene glycol precipitation method (Arch. Virol., 71, 185 (1982)), the filter concentration method (J. Cell. Biol., 111, 217 (1990)), etc.